We are pursuing a replication-competent Adenovirus (Ad)-recombinant priming/subunit boosting approach as a prophylactic vaccine for HIV/AIDS. Studies showing that the vaccine regimen can elicit humoral, cellular, and mucosal immunity together with significant protection in non-human primate models has resulted in movement of this approach towards Phase I human trials. At the same time, further pre-clinical studies are evaluating the utility of recombinants encoding additional viral genes, investigating novel subunit protein boosts, and exploring routes of immunization for elicitation of the best overall immune responses and protective efficacy. A recent study investigated complex Ad5hr-SIV recombinant priming regimens, including one encoding a non-myristoylated SIV nef, together with boosting by either gp120 or a peptide polymer representing the CD4 binding site of the envelope protein. Impressive results were obtained which showed that 39% of macaques immunized with recombinants encoding 3 or more SIV genes and boosted with gp120, or immunized with the Ad5hr-SIVenv/rev recombinant only but boosted with the peptide polymer, were highly protected. Following challenge with the highly pathogenic SIVmac251 isolate, viremia was either undetectable or it was cleared or controlled at the threshold of detection. Cellular immune responses were significantly correlated with control of viremia during the chronic phase of infection, while anti-envelope binding antibodies were significantly correlated with reduced acute phase viremia. These antibodies were subsequently shown to mediate antibody-dependent cellular cytotoxicity (ADCC). Continuing studies are investigating the role of ADCC in protection against HIV replication and disease progression. In follow-up studies, 73% of the highly protected macaques continued to exhibit potent protection against a second challenge with SIVmac251 administered one year later with no intervening immunization. This durable protection was associated with cellular immune responses as shown by in vivo depletion of CD8+ T cells. In the chimpanzee model, we have shown that mucosal immunization with replication-competent Ad-HIVenv recombinants elicit enhanced cellular immune responses and better prime antibody responses compared to non-replicating Ad-recombinants containing the same inserted HIV gene. We believe the replication competent vector may be most advantageous for vaccine design, as it can achieve equivalent expression of inserted genes at lower doses, can more effectively target mucosal inductive sites, can better elicit cytokines and co-stimulatory molecules which serve as natural adjuvants, and has a greater potential for generating more persistent immunity. Investigation of novel HIVnef immunogens has shown that Ad-recombinants encoding non-myristoylated nef elicit more potent cellular immune responses compared to wild-type nef. Removal of the myristoylation site prevents down-modulation of both MHC-class I molecules and CD4 from the surface of cells expressing the protein, a mechanism by which HIV can avoid a strong host cellular immune response. Finally, Ad-recombinants encoding HIV wild-type tat or a transdominant tat mutant were shown to be immunogenic in mice. Importantly, immunization with either Ad-HIVtat recombinant resulted in enhanced cellular immune responses to a co-administered SIV Gag antigen. On-going studies are investigating the immunogenicity and protective efficacy of these tat recombinants in non-human primates.